Field of the Invention
This invention relates to bacteria detection such as bacterial viability and drug resistance through metabolic monitoring, and in particular, it relates to bacteria detection using oxygen sensitive fluorescent materials and methods for enhancing co-localization of the bacteria and the fluorescent materials.
Description of Related Art
A rapid bacteria detection technology using micro-well array and oxygen sensitive fluorescent film is described in Ayyash et al., Fast and inexpensive detection of bacterial viability and drug resistance through metabolic monitoring, 2014 Health Innovations and Point-of-Care Technologies Conference (Seattle, Wash. USA) Oct. 8-10, 2014 (“Ayyash et al. 2014”). In this technology, oxygen consumption in the well including the bacteria leads to fluorescent signal emission. Excerpts of the paper are presented below:                [We introduce] an innovative detection method to produce rapid and accurate diagnosis of bacterial infection through miniaturization and parallelization. This method is demonstrated with wells of several shapes (square, circle), diameters (100-1000 μm) and depths (≤100 μm). In the development of proof of concept, we use laboratory strain of E. coli as the model pathogen. The integration of the fluorescent oxygen sensor, ruthenium tris (2,2′-diprydl) dichloride hexahydrate (RTDP), allows us to monitor the dissolved oxygen concentration as a measure of bacterial metabolism. Detection time of the bacteria within the microwells can be as fast as a few of hours (4-5 hrs), with concentrations that vary between 102 to 108 cells/mL. Adding the appropriate drug to the broth and measuring growth through fluorescence also probed drug resistance. This reported method for microfabrication of the wells, is rapid, economical, versatile and simple. (Abstract.)        In this method, the sample is placed in a chamber with a growth medium that is specific for the particular bacteria of interest. This liquid medium provides a specific condition for the growth of the specific bacteria of interest while preventing other contaminating species from growing. A fluorophore that is quenched in the presence of oxygen is dissolved in the medium. Since bacteria are aerobic, they consume oxygen that is present in the medium during metabolism and depletes the oxygen in the surrounding environment, producing fluorescence. Drug resistance can be probed by adding the appropriate drug to the broth and measuring growth or lack of it through fluorescence. (Pp. 22-23.)        When this metabolic monitoring is done in large volumes (1-10 mL) it still takes a long time. However if the sample were segmented into thousands of smaller volumes then some of the wells will contain the bacteria of interest while others will not. The process of segmentation will increase the local concentration of the bacteria by several orders of magnitude. Therefore the nutrients present in that small volume will be quickly depleted and that event can be sensed faster. This is the working principle behind our fast metabolic monitoring of bacteria. (P. 23.)        Experimental Setup and Procedure. In a typical experiment, the sample is mixed with a solution containing the growth medium (Luria-Bertani (LB) medium) and an oxygen sensitive fluorophore (ruthenium tris(2,2′-diprydl) dichloride hexahydrate, (RTDP)-0.1 mg/mL) and dispensed on to the microarray. A simple swiping process dispenses the sample into the hydrophilic microwells while the hydrophobic top surface removes the sample from the top cleanly (step 4,5 in FIG. 1). Next, the microarray is capped using a glass slide (made hydrophobic using a surfactant) and imaged under a fluorescent microscope to measure the intensity of the fluorophore. (P. 23.)        
Part of FIG. 1 of Ayyash et al. 2014 is reproduced in FIG. 1 of the instant application. FIG. 1A of the instant application schematically illustrates an example of segmentation described in Ayyash et al. 2014, which increases the local concentration of the bacteria and oxygen is depleted quickly.